1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to methods for inducing controlled cell death or apoptosis in a eukaryotic cell by providing either DNA encoding the Shigella IpaB protein or the IpaB protein to the cell. This method is useful in treating diseases or disorders treatable by the eradication of unwanted cells, for example cancer, autoimmunity, inflammation and chronic viral infections.
2. Description of the Background Art
Apoptosis, or programmed cell death, is considered an essential process in normal development of multicellular organisms. Apoptosis is also thought to serve as a defense against viral infection and oncogenesis (Thompson, C. B., Science 267:1456-1462 (1995)).
A number of activators or inducers of apoptosis are listed in Table I, below. (See also Thompson, supra.). Diseases which are associated with either induction or inhibition of apoptosis are listed in Table II, below.
Cellular and certain viral genes act as inhibitors of apoptosis. The best known cellular "death repressor" gene, bcl2, was first discovered in human tumor cells. This gene can replace the structurally similar adenoviral elb gene (Rao, L. et al., Proc. Natl. Acad. Sci. USA 89:7742 (1992)) as an inhibitor of apoptosis (Boyd, J. M. et al., Cell 79:341 (1994)). Other genes which are similar in sequence and function to bcl2 are the bhrf1 gene of Epstein-Barr virus (EBV) and the lmw5-hl gene of African swine fever virus (Neilan, J. G., et al, J. Virol. 67:4391 (1993), Henderson, S. et al., Proc. Natl. Acad. Sci. USA 90:8479 (1993)). Other structurally dissimilar genes that inhibit
TABLE I ______________________________________ INDUCERS OF APOPTOSIS Damage-Related Inducers, Drugs Physiological Activators and Toxins ______________________________________ TNF family (Fas ligand, TNF) Viral infections, Bacterial toxins, TGF.beta. Oncogenes (myc, rel, E1A) Neurotransmitters (glutamate, Tumor Suppressors (p53) dopamine, NMDA) Cytotoxic T lymphocytes Withdrawal of growth factors, Oxidants and free radicals nutrient deprivation Heat shock Loss of matrix attachment Chemotherapeutic drugs Calcium (antimetabolites) Glucocorticoids Gamma and UV irradiation Ethanol .beta.-amyloid peptides ______________________________________
TABLE II ______________________________________ Diseases Associated with Induction or Inhibition of Apoptotic Cell Death Inhibition of Apoptosis Increased Apoptosis ______________________________________ 1. Cancer 1. AIDS Carcinomas with p53 mutations 2. Neurodegenerative disorders Follicular Alzheimer's disease lymphomas Parkinson's disease Hormone-dependent tumors Amyotrophic lateral sclerosis Breast cancer Retinitis pigmentosa Prostate cancer Cerebellar degeneration Ovarian cancer 3. Myelodysplastic syndromes 2. Autoimmune disorders Aplastic anemia Systemic lupus erythematosus 4. Ischemic injury Immune glomerulonephritis Myocardial infarction 3. Viral infections Stroke Herpesviruses Reperfusion injury Poxviruses 5. Toxin-induced liver diseases Adenoviruses Alcoholism ______________________________________
apoptosis include the p35 gene and the iap gene in baculoviruses ((Clem, R. J. et al., Science 254:1388 (1991); Molec. Cell Biol. 14:5212 (1994)); cowpox virus crmA (Ray, C. A. et al., Cell 69:597 91992)), Herpesvirus K1.34.5.
A central death effector molecule in the apoptosis pathway is the cysteine protease interleukin-1.beta.-converting enzyme (ICE). This enzyme was first discovered based on its cleavage of IL-1.beta. precursor protein to mature active IL-1.beta.. ICE therefore plays a crucial role in the initiation of cytokine cascades involved in inflammatory and host defense responses. ICE is closely related to the protein encoded by the Caenorhabditis elegans cell death gene, ced-3, the product of which is required for cells to undergo programmed cell death during development (M. Miura et al., Cell 75:653 (1993)). The important role of ICE in apoptosis is supported by the observations that a number of the viral inhibitors of apoptosis are specific inhibitors of ICE.
Recent evidence suggests that the failure of cells to undergo apoptotic cell death might be involved in the pathogenesis of a variety of human diseases, including cancer, autoimmune diseases, and viral infections (Vaux, G. et al., Cell 76:777 (1994); Bursch, W. et al., Trends Pharmacol Sci. 13:245 (1992)). In contrast, a large number of diseases characterized by cell loss, including neurodegenerative disorders, AIDS (acquired immunodeficiency syndrome), and osteoporosis, may result from accelerated rates of physiologic cell death. Hence, the art recognizes the need for specific methods designed to enhance or decrease the susceptibility of individual cell types to apoptosis as a basis for treating a variety of human diseases. It is to this problem that the present invention is addressed.
Apoptosis Induced by Shigella Bacteria
Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. After being phagocytosed by a macrophage, Shigella flexneri cells escape from the phagosome into the cytoplasm and induce apoptosis both in vitro (Zychlinsky, A. et al., Nature 358:167-168 (1992)) and in vivo. S. flexneri invasiveness and cytotoxicity are encoded in a 220 kb plasmid (LaBrec, E. H. et al., J. Bacteriol. 88:1503-1518 (1964)) which includes among many other genes the ipa operon. The gene products IpaB, C and D are essential for cell invasion (Menard, R. et al., J. Bacteriol. 175:5899-5906 (1993); High, N. et al., EMBO J. 12:1191-1999(1992)). However, Ipab is required to initiate apoptosis. This was shown by using an ipaB deletion mutant which lacked invasiveness (Zychlinsky, A., et al. Molec. Microbiol. 11:619-627 (1994)). In the absence of ipaB, apoptosis does not occur. IpaB and its neighboring genes of the Shigella invasion plasmid have been cloned and characterized (Buysse, J. M. et al., J. Bacteriol. 169:2561-2569 (1987; Baudry, B. et al., J. Gen. Microbiol. 133:3403-3413 (1987); Baudry, B. et al., Microb. Pathogenesis 4:345-357 (1988)). The nucleotide sequence (SEQ ID NO:1) of ipaB and the amino acid sequence of the IpaB protein (SEQ ID NO:2) are provided below. However, the ability of IpaB alone to induce apoptosis upon delivery to a target cell as an isolated gene or protein has not previously been known and is the subject matter of the present invention.
Homologues of IpaB have been found in other bacterial species. These include yopB of Yersinia enterocolitica and Yersinia pseudotuberculosis (Hakansson, S. et al., Infect. Immun. 61:71-80 (1993)), sipB in Salmonella typhimurium (Kaniga, K. et al., J. Bacteriol 177:3965-3971 (1995) and in Salmonella typhi (Hermant, D. et al., Mol. Microbial 17:781-789 (1995)). The complete nucleotide sequences encoding these four IpaB homologues are SEQ ID NO:3, 5, 7 and 9, respectively. The amino acid sequences of these four homologues are SEQ ID NO:4, 6, 8 and 10, respectively.